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EP0127841B1 - Methode zum Färben mit Silbersalzen - Google Patents

Methode zum Färben mit Silbersalzen Download PDF

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Publication number
EP0127841B1
EP0127841B1 EP84105847A EP84105847A EP0127841B1 EP 0127841 B1 EP0127841 B1 EP 0127841B1 EP 84105847 A EP84105847 A EP 84105847A EP 84105847 A EP84105847 A EP 84105847A EP 0127841 B1 EP0127841 B1 EP 0127841B1
Authority
EP
European Patent Office
Prior art keywords
solution
silver
silver staining
molecular weight
polyethylene glycol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP84105847A
Other languages
English (en)
French (fr)
Other versions
EP0127841A1 (de
Inventor
Kazuaki Oosawa
Nobuyoshi C/O Tokyo Laboratory Of Ebata
Miyoshi C/O Tokyo Laboratory Of Hirata
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Daiichi Pure Chemicals Co Ltd
Original Assignee
Daiichi Pure Chemicals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Daiichi Pure Chemicals Co Ltd filed Critical Daiichi Pure Chemicals Co Ltd
Priority to AT84105847T priority Critical patent/ATE36195T1/de
Publication of EP0127841A1 publication Critical patent/EP0127841A1/de
Application granted granted Critical
Publication of EP0127841B1 publication Critical patent/EP0127841B1/de
Expired legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Definitions

  • This invention relates to silver stain and more particularly, to a method for staining a carrier, which bears thereon substances being detected, with silver in which the carrier is pretreated with an neutral or approximately neutral alcoholic solution comprising polyethylene glycol or a polyoxyethylene alkylphenol and subsequently treated with a silver staining solution of a specific type.
  • a silver staining method comprising the following steps:
  • a sole figure is a band image obtained in Example 3 in which indicated by a is a band image of BSA, by 1, 2, 3, 4, 5 and 6 are respectively, images obtained by electrophoresis of 5 ng, 1 ng, 500 pg, 100 pg, 50 pg and 10 pg of BSA.
  • a carrier which has been subjected to electrophoresis by a usual manner (hereinafter referred to simply as gels) is immersed in an alcoholic solution containing polyethylene glycol or a polyoxyethylene alkylphenol (hereinafter referred to as washing and fixing solution) for about 10 to 60 minutes, preferably about 25 minutes, thereby causing the gels to be shrunk. By the shrinkage, the water and the reagent are released from the gels.
  • the alcohols used in the washing and fixing solution include lower alcohols and preferably linear or branched alcohols having from 1 to 4 carbon atoms.
  • linear or branched alcohols examples include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and mixtures thereof. Of these, isopropanol, or a mixture of methanol and ethanol is preferred. These alcohols are conveniently used as an aqueous solution having a concentration of from 30 to 70 (v/v)%, preferably about 50 (v/v)%.
  • Polyethylene glycol suitable for the purpose of the invention has an average molecular weight of from 600 to 3500, preferably 1500 to 2500.
  • the polyoxyethylene alkylphenol is represented by the formula, in which R' represents a linear or branched alkyl having from 6 to 10 carbon atoms, and n is an integer of 7 to 10 which indicates the center of the distribution.
  • R' is octyl and n which indicates a center of the distribution is 9.
  • the polyethylene glycol (PEG) or polyoxyethylene alkylphenol is generally used in an concentration of 0.05 to 5 (w/v)%, preferably about 2 (w/v)%, of the washing and fixing solution.
  • the gels pretreated with the washing and fixing solution are subsequently immersed in and treated with a silver staining solution for 10 to 30 minutes, preferably about 15 minutes.
  • the silver staining solution used for the above treatment is a mixture of silver nitrate, a compound of the formula R-NH 2 (R-NH 2 has the same meaning as defined before), and a caustic alkali in mixing ratios by molecular weight of 1:4.5-9.5:1.0-26.0.
  • the treatment with the silver staining solution within the above mixing ratios permits staining in good reproducibility and shortening of the operation time.
  • Compounds of the formula, R-NH 2 usable in the present invention include; for example, ammonia, ethanolamine, propanolamine, isopropanolamine, butanolamine, isobutanolamine, tris(hydroxymethyl)aminomethane, methylamine, ethylamine, propylamine, butylamine, isobutylamine, etc.
  • Examples of the caustic alkali include sodium hydroxide, potassium hydroxide, etc.
  • the gels treated with the silver staining solution is then immersed in a developer solution for about 10 to 15 minutes. If necessary, after treatment with the developer, it may be further immersed in a solution of 0.5 to 2 (w/v)% chloroauric acid, by which a clearer image is obtained.
  • the method of the present invention is conveniently applied to polymerized gels having the network structure, such as polyacrylamide gel, agar-agar, agarose, etc.
  • the method of the invention ensures formation of a clear image in about 60 minutes. Moreover, the same procedure is applied to any substances contained in gels such as proteins, nucleic acids, sugars, lipids, etc.
  • the method of the invention has the following features:
  • Silver nitrate, ammonia and sodium hydroxide were dissolved in distilled water in ratios by molecular weight of 1:7:4.3 to make a total volume of 200 ml.
  • citric acid 0.05% citric acid, 37% formalin and distilled water were used to make 200 ml of a 0.02 v/v% formalin solution of 0.005 w/v% citric acid.
  • the evaluation standard is the same as in Example 1.
  • SDS-PAG sodium dodecylsulfate- polyacrylamide gel
  • Isopropanol and polyethylene glycol 2000 were diluted with distilled water to make a total volume of 200 ml with concentrations of 50% (v/v) and 1 % (w/v), respectively.
  • Silver nitrate, ammonia and sodium hydroxide were dissolved in distilled water to have mixing ratios by molecular weight of 1:4.8:1. The total volume was brought up to 200 ml.
  • Reagents (1), (2) and (3) were, respectively, placed in pan of the cabinet size.
  • the sample was first immersed in the pan of reagent (1) for 25 minutes and removed from the pan, followed by immersing in the reagent (2) thereby causing the silver staining solution to penetrate into the gel.
  • the sample was immersed in a pan containing 200 ml of distilled water and washed. This procedure was repeated three times. Finally, the washed sample was immersed in the reagent (3) for 15 minutes to form a brown to black band image.
  • Methanol, ethanol and polyethylene glycol 2000 were diluted with distilled water to make a total volume of 200 ml with concentrations of 40% (v/v), 10% (v/v) and 2% (w/v), respectively.
  • Silver nitrate, ammonia and sodium hydroxide were dissolved in distilled water to have mixing ratios by molecular weight of 1:7:4.3. The total volume was brought up to 200 ml.
  • Example 3 The operation of Example 3 was repeated using the reagents indicated above, thereby obtaining band image similar to the image of Example 3..
  • Isopropanol and polyoxyethylene phenol (commercial name of Rohm and Haas Co., Ltd., of U.S.A.: Nonidef S P-40) were diluted with distilled water in concentrations of 50% (v/v) and 3% (v)/v) to make a total volume of 200 ml.
  • Silver nitrate, ammonia and sodium hydroxide were dissolved in ratios by molecular weight of 1:9.1:25.5 to make a total volume of 200 ml.
  • Example 3 The procedure of Example 3 was repeated using the reagents indicated above, thereby obtaining band image similar to the image of Example 3.
  • Silver nitrate, tris(hydroxymethyl)aminomethane and sodium hydroxide were dissolved in distilled water in ratios by molecular weight of 1:5:1.0 to make a total volume of 200 ml.
  • Example 3 The procedure of Example 3 was repeated using the reagents indicated above, thereby obtaining a band image similar to the image of Example 3.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Claims (1)

  1. Methode zum Anfärben mit Silber, umfassend die folgenden Stufen:
    (1) Vorbehandlung eines Trägers, auf dem eine zu ermittelnde Substanz aufgebracht ist, mit einer Lösung von niederen Alkoholen oder einer Mischung von niederen Alkoholen mit 1 bis 4 Kohlenstoffatomen, wobei die Lösung 0,01-5 Gew.Nol.% Polyäthylenglycol oder Polyoxyäthylenalkylphenol (wobei das Alkyl ein Alkyl mit 6 bis 10 Kohlenstoffatomen bedeutet) enthält, und
    (2) Behandlung des vorbehandelten Trägers mit einer Lösung, umfassend Silbernitrat, Ammoniak oder ein primäres Amin und Ätzalkali in Verhältnissen, ausgedrückt durch das Molekulargewicht von 1:4,5-9.5-1,0-26,0, und
    (3) Waschen mit Wasser, und anschließend
    (4) Entwicklung in einer Entwicklerlösung.
EP84105847A 1983-05-26 1984-05-22 Methode zum Färben mit Silbersalzen Expired EP0127841B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT84105847T ATE36195T1 (de) 1983-05-26 1984-05-22 Methode zum faerben mit silbersalzen.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP58091487A JPS59217164A (ja) 1983-05-26 1983-05-26 銀染色方法
JP91487/83 1983-05-26

Publications (2)

Publication Number Publication Date
EP0127841A1 EP0127841A1 (de) 1984-12-12
EP0127841B1 true EP0127841B1 (de) 1988-08-03

Family

ID=14027765

Family Applications (1)

Application Number Title Priority Date Filing Date
EP84105847A Expired EP0127841B1 (de) 1983-05-26 1984-05-22 Methode zum Färben mit Silbersalzen

Country Status (5)

Country Link
US (1) US4582808A (de)
EP (1) EP0127841B1 (de)
JP (1) JPS59217164A (de)
AT (1) ATE36195T1 (de)
DE (1) DE3473177D1 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104458388A (zh) * 2014-12-01 2015-03-25 中国农业科学院棉花研究所 一种page的高效银染方法

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5064768A (en) * 1988-09-16 1991-11-12 Daiichi Pure Chemicals Co., Ltd. Silver staining technique and kit
US6074818A (en) * 1990-08-24 2000-06-13 The University Of Tennessee Research Corporation Fingerprinting of nucleic acids, products and methods
WO1992017611A1 (en) * 1991-03-28 1992-10-15 The University Of Tennessee Research Corporation Dna silver staining
US5795785A (en) * 1992-12-10 1998-08-18 Suslov; Eugene Ivanovich Method for the diagnosis of human malignant tumors
US5595878A (en) * 1995-06-02 1997-01-21 Boron Biologicals, Inc. Detection of biopolymers and biooligomers with boron hydride labels
EP1344055A4 (de) * 2000-11-20 2009-03-25 Invitrogen Corp Materialien zur verbesserung der anfärbung von biopolymeren in matrizes
US7498140B2 (en) * 2001-03-08 2009-03-03 Pierce Biotechnology, Inc. Direct detection of biomolecules in a gel
WO2002073157A2 (en) * 2001-03-08 2002-09-19 Pierce Biotechnology, Inc. Direct detection of biomolecules in polyacrylamide gel
WO2002078906A2 (en) * 2001-03-29 2002-10-10 Cellect Technologies Corp. Methods devices and systems for sorting and separating particles
CN111841496B (zh) * 2020-07-24 2022-02-22 泉州南京大学环保产业研究院 一种快速制备载银活性炭的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3997656A (en) * 1973-06-25 1976-12-14 Applied Bioscience Tissue staining method and composition
US4416998A (en) * 1981-04-02 1983-11-22 The Upjohn Company Silver stain procedure and kit for same
US4468466A (en) * 1982-02-16 1984-08-28 The United States Of America As Represented By The Department Of Health And Human Services Silver stains for protein in gels--a modified procedure

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Electrophoresis Vol. 2 No. 1, February 1981 pages 141-147 H.M. Pochling, V.Neuhoff "Visualization of proteins with a silver "stain": A critical analysis" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104458388A (zh) * 2014-12-01 2015-03-25 中国农业科学院棉花研究所 一种page的高效银染方法

Also Published As

Publication number Publication date
JPS59217164A (ja) 1984-12-07
EP0127841A1 (de) 1984-12-12
ATE36195T1 (de) 1988-08-15
DE3473177D1 (en) 1988-09-08
JPH0129423B2 (de) 1989-06-09
US4582808A (en) 1986-04-15

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